Project Summary Cilia and flagella are conserved microtubule-based cell extensions present on most cells in the mammalian body. In addition to their role in cell locomotion and fluid transport, cilia participate in cellular sensing and signaling. Over the past two decades, it has been established that numerous developmental anomalies and diseases are caused by dysfunctional cilia. The goal of our work is to understand how cells assemble and maintain cilia, which both require protein transfer between the cell body and the organelle. A key mechanism that determines the protein content of cilia is intraflagellar transport (IFT), a motor-based motility of large carriers (?IFT trains?) that move proteins in and out of cilia. We will use Chlamydomonas reinhardtii as a unicellular model to determine how IFT identifies proteins destined for the cilium and how the cells regulate the volume and timing of ciliary protein traffic. In Aim1, we will focus on the transport of tubulin, the main structural protein of cilia and flagella. The amount of tubulin and other axonemal proteins entering cilia on IFT trains is upregulated while cilia grow. Tubulin also enters cilia by diffusion and we will establish the quantitative contribution of each route in cilia assembly. We will determine if IFT54 is part of the previously characterized IFT74-IFT81 tubulin-binding module or if it forms an independent tubulin-binding site. All three proteins interact with tubulin via their tubulin-binding domains (TBDs). Isolated IFT complexes will be used to study if the TBDs undergo biochemical changes related to cargo binding and cilia length. We will attempt to map the TBDs on isolated IFT particles and we will study whether IFT particles undergo structural changes inside cilia potentially explaining the differences in cargo binding. We expect to gain insights into how cells regulate tubulin transport, which is critical for the timing of ciliogenesis and the regulation of ciliary length. In Aim 2, we will focus on the transport of proteins associated to the ciliary membrane by lipidation. Such proteins are critical for the sensory and signaling functions of cilia. Often, they enter and exit cilia to modulate signaling but the role of IFT in this traffic is mostly unknown. In cilia of C. reinhardtii mutants in BBS proteins or Arl13b, the patterns of membrane-associated proteins are severely affected. In humans, mutations in BBS proteins and Arl13b result in Bardet-Biedl syndrome (BBS) and Joubert syndrome, respectively. Both mutants show loss and abnormal accumulation of membrane-associated proteins in cilia, raising the question whether more than one route of transport is affected. We will use in vivo imaging to determine the role of IFT and diffusion in ciliary entry and export of proteins mislocalized in these mutants. We will test a hypothesis that initial ciliary defects caused directly by the bbs and arl13b mutations will induce additional biochemical defects increasingly impairing cilia over time.